The detection of carriers of deleterious, mutant alleles is very useful medically to guide the clinical treatment of a patient. That is, it is valuable to know whether a person has a germline or an acquired mutation associated with a disease or susceptibility to a disease.
For example, pre-operatively identifying a colon cancer patient who carries a germline mutation in a mismatch repair gene associated with hereditary non-polyposis colon cancer (HNPCC) guides a surgeon in deciding whether or not to perform a total colectomy or a partial colectomy. If the patient has a germline mutation, then the chance for a second primary colon cancer is extremely high (perhaps 70%). In that case, a total colectomy is usually recommended as the initial surgical treatment. The objectives of the surgery would be to treat the colon cancer that already exists and to prevent the development of new colonic malignancies. The presence of a germline mutation in a cancer patient will not only lead to differences in the surgical treatment of the cancer, but also to possible differences in the need for adjuvant chemotherapy after colectomy.
The ability to identify the presence of a germline mutation in patients will also lead to more effective approaches toward cancer prevention and early detection of other cancer types (e.g., besides colon cancer) that such patients are at risk to develop. In other words, the information from a test that identifies HNPCC-affected colon cancer patients should trigger a second clinical screen of those patients for extracolonic cancers which could also be life-threatening.
In addition, positive diagnoses of germline mutations in cancer patients will facilitate testing for detection of germline mutation carriers in their family members. Such testing will lead to more effective cancer prevention strategies and to better clinical management for cancers that are detected in family members. Thus, the information provided by this invention will also be useful to the physician in recommending the screening of other members of the patient's family since the finding of a germline mutation puts those individuals at high risk for colon cancer and other cancers.
A major problem in the diagnosis/prognosis of hereditary diseases is the enormous amount of work and cost involved in performing current molecular based assays to identify hereditary traits associated with a disease, such as HNPCC, or disease susceptibility. The instant invention provides more feasible methods, immunoassays, to detect such hereditary traits. The assays of this invention are relatively simple, rapid (24 hour turnaround), high throughput and inexpensive. The assays of this invention make feasible the screening of entire populations at risk for such hereditary diseases. Also, the assays of this invention can identify carriers of disease-associated hereditary traits before they develop a disease, such as cancer.
The assays of this invention are based on the assumption that gene expression directly relates to gene dosage, that is, the presence of two wild-type alleles will result in the expression of twice the amount of full-length wild-type protein than would occur if only one wild-type allele were present. In accordance with this invention, immunoassays are used to measure a reduction from normal in the amount of full-length protein expressed by a subject gene. In contrast, mutations in genes, such as mismatch repair (MMR) genes, are classically detected by DNA sequence analysis and/or in vitro translation-type assays [Giardiello, F. M., “Genetic Testing in Hereditary Colon Cancer,” JAMA, 278: 1278-1281 (1997)], tests which are costly, time consuming and only offered in select academic and commercial reference labs.
Representative immunoassays are disclosed which are used to screen primarily for certain types of hereditary colorectal cancer (CRC) or a predisposition to hereditary CRC. Such representative immunoassay methods to screen for hereditary CRC or a predisposition thereto are based on the detection of cellular full-length protein level changes that are due to either (1) mutations of the adenomatous polyposis gene (APC), a mutation that is associated with familial adenomatous polyposis (FAP), or to (2) mutations of mismatch repair (MMR) genes, particularly, MLH1, MSH2, PMS1, PMS2, and MSH6, mutations that are associated with hereditary non-polyposis colon cancer (HNPCC).
Molecular genetic methods, such as those based on the detection of mutations in the DNA sequence of an allele of a gene of interest, have been used to diagnose individuals carrying hereditary colon cancer traits such as HNPCC and FAP. Although such tests can be highly specific and are fairly easy to perform when the precise target mutation is known, e.g., when screening HNPCC kindreds, such tests are difficult to perform, when the precise target mutation is not known. In the latter case, finding a small DNA mutation among a panel of large MMR genes becomes daunting, akin to finding a needle in a haystack. It is precisely in this situation that the immunoassays of this invention become so practical because they are rapid and relatively inexpensive.
Further, a positive finding in the immunoassay testing of this invention can be confirmed by DNA sequencing in a small fraction of the time that would have been necessary had DNA tests alone been used. Thus, the immunoassays of this invention may become useful as a complementary “pre-test”. This invention may also support molecular genetic tests in the diagnosis of affected members of known kindreds in which the mutation has already been identified.
As indicated above, unknown mutations are very difficult to detect by molecular genetic tests. Unknown mutations are those found in probands, isolated cases and kindreds not yet studied. The germline mutations in such populations, which mainly involve single base pair changes, small insertions, and small deletions, may be distributed over a large portion of a gene and may involve any one of several possible loci. Moreover, the large size of the coding region makes identification of mutations by gene sequencing very difficult, labor intensive, and costly. Therefore, other techniques have been developed and are used to detect the presence of DNA variations and mutations. Such techniques include denaturation gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP) analysis and RNAase protection analysis. However, each of those other approaches also has drawbacks and/or limitations, particularly in sensitivity. For example, the sensitivity of those techniques for detecting APC mutations in FAP patients is only 30-70%, depending upon the method used. Newer techniques have been developed to detect mutations by analyzing translation products synthesized in vitro, but only have a slightly better sensitivity.
Most of the molecular genetic approaches discussed above are designed to detect single base pair changes, small insertions, and small deletions and/or mutations that lead to termination of translation. However, in some instances, deletion of the entire gene may occur in the germline of patients. This is yet another drawback of earlier methods because deletions of the entire gene are missed by molecular genetic approaches. Furthermore, some patients may have promoter or splicing mutations that lead to reduced levels of normal transcripts, i.e., mutations located outside the coding region that is analyzed by most molecular methods. In contrast, the immunoassay methods of this invention, by detecting the level of wild-type protein, may have greater sensitivity than the molecular genetic approaches, since they are able to detect, in addition to standard mutations, the mutations involving allelic loss, and mutations in the promoter, enhancer and splice site regions.
Another disadvantage of current molecular genetic techniques is that the technology of molecular genetics is not established in most pathology laboratories. Also, the results of molecular genetic assays often cannot be obtained quickly, for example, prior to surgery. Although molecular genetic tests can be performed on cancer tissue samples, the question whether the mutation in the cancer cells was acquired or germline would not be answered.
For example, one approach currently being explored is the detection of microsatellite instability (i.e., mutations in repetitive DNA sequences) in tumor specimens, rather than detection of specific MMR mutations [Dietmaier et al., “Diagnostic microsatellite instability: Definition and correlation with mismatch repair protein expression,” Cancer Res., 57: 4749-4756 (1997)]. The microsatellite approach involves molecular biologic techniques (PCR or Southern Blot analysis) to screen for genetic changes in a panel of different genetic loci (usually 5 or more genes are simultaneously analyzed for microsatellite instability). The microsatellite approach is fairly easily performed by a molecular geneticist, and the aim is to identify which polyps have arisen from a MMR mutation. Again, however, this approach does not distinguish between acquired and germline MMR mutations and provides no information that identifies which of the five known MMR genes is mutant.
The instant immunoassay methods, in contrast, can be done in any pathology laboratory and can be developed to be cost-effective to screen large numbers of individuals in a short amount of time. The assays can be performed quickly, and the results are immediately obtainable. Once the change in the product of a particular gene is identified by the immunoassay methods of the invention, molecular genetic tests can then be employed to determine the precise location of the mutation.